2.4. Cell Viability on the Decellularized Matrix

To examine whether the DEM supports cell growth of esophageal cancer cells, an esophageal cancer cell line, KYSE30 from Sigma-Aldrich (St. Louis, MO, USA), was used. KYSE30 was cultured in RPMI 1640 Medium (1× Gibco, ref A10491-01) with antibiotics/mycotic (Gibco, streptomycin sulfate and Penicillin G sodium/Amphotericin B, ref 15240-062) at 37 °C in a CO₂ incubator. When cells were grown to near confluence, they were trypsinized and cell suspensions were seeded on the DEM under two culture conditions: static culture and dynamic culture. For static conditions, DEM was placed in 24-well plate and seeded with 1 × 10⁵ KYSE30 cancer cells, and media was changed every 2 days. For dynamic conditions, a 40 µL cell suspension of around 1 × 10⁵ KYSE30 cells was seeded onto the inner lumen surface of the DEM circumference. After cell seeding, DEM was rested for 2 h to allow time for cells to attach and subsequently mounted to circulation jars where culture medium was continually perfused at 15 µL/minute for the remaining duration of experiment. For the dynamic culture system, typical 1000 µL pipette tips were cut to desired lumen diameter and fit for flow orifice, then perforated using a razor blade to allow cell seeding from the sides into lumen (cut ‘windows’ into the pipette tip for cell seeding and diffusion characteristic), and flow/diffusion of culture media to extraneous feature of flow chamber. Solution passively diffused through the perforations and applied flow rate through orifice condition allows consistent nutrition and waste removal, thus enabling dynamic culture conditions with variable flow rate and as needed diameter setting, which accounted for variability in esophageal matrix.

To verify the cell viability of KYSE30 cultured on the DEM, after 7 days, a Live/Dead^® Viability/Cytotoxicity Kit for mammalian cells from Invitrogen was conducted. Cells/DEMs were stained with 6 μM EthD-1 and 2 μM Calcein AM and incubated at room temperature for 40 min in the dark, and then washed and stored in PBS before analysis by microscopy. Red indicates dead cells and green indicates alive cells.